Nanoparticulate cell culture surface

ABSTRACT

A cell culture article including a substrate having nanoparticles on the substrate surface, the nanoparticle including:
         a polymer of formula (I)       

     
       
         
         
             
             
         
       
     
     where (x), (y), (z), R, R′, R″, S, W, and X, are as defined herein. Methods for making the cell culture article or cell culture article and methods for performing an assay of a ligand with the article are also disclosed.

CROSS-REFERENCE TO RELATED CO-PENDING APPLICATIONS

This application claims the benefit of European Application Serial No.08305846.1, filed Nov. 26, 2008, entitled NANOPARTICULATE AFFINITYCAPTURE FOR LABEL INDEPENDENT DETECTION SYSTEM and is related toco-pending U.S. patent application Ser. No. 12/620,100, filed Nov. 17,2009 having the same title. The entire disclosure of publications,patents, and patent documents mentioned herein are incorporated byreference.

BACKGROUND

The disclosure generally relates to surfaces for cell culture, and moreparticularly to surface chemistry for cell culture in the presence ofand absence of serum in cell culture media.

SUMMARY

The disclosure provides surface treated articles for cell culture, andmethods for their preparation and use. The cell culture surfaces providesubstrates for the culture of cells in the presence of and absence ofserum as well as properties amenable for biosensors for labelindependent detection (LID) and high efficiency biosensors such as Epic®biosensors, resonant grating and like sensing applications. Thedisclosure also concerns systems and methods providing sensors capableof immobilizing bioentities (e.g., receptor proteins, and like cellulartargets) at high density and providing superior sensitivity with respectto detecting an analyte than previously reported. The LID biosensors ofthe disclosure have higher sensitivity for the detection ofbio-molecular recognition events. The treated biosensor surfaces of thedisclosure can exhibit increased ligand binding, consume less protein,and provide greater sensitivity compared to known treated biosensorsurfaces. The treated biosensor surfaces of the disclosure are alsosuitable for cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show exemplary polymer formulas comprising nanoparticles andthere function in affinity capture, in embodiments of the disclosure.

FIGS. 2A-C show SEM images of NTA derivatized EMA nanoparticles, inembodiments of the disclosure.

FIG. 3 shows SPR responses for the surfaces with and withoutnanoparticle texture of Example 2, in embodiments of the disclosure.

FIG. 4 shows SPR responses using a commercially available NTA chipwithout nanoparticle texture compared with the present Example 3 withnanoparticle texture, in embodiments of the disclosure.

FIG. 5 shows a comparison of His-tag CAII immobilization levels on anaffinity surface only and on the affinity/covalent surface of Example 4,in embodiments of the disclosure.

FIG. 6 shows a comparison of protein leaching responses on the affinitysurface and the affinity/covalent surface of Example 4, in embodimentsof the disclosure.

FIG. 7 shows a comparison of binding values on the affinity surface onlyand on the affinity/covalent surface of Example 4, in embodiments of thedisclosure.

FIG. 8 shows a comparison of His-tag CAII immobilization levels on theaffinity surface and on the affinity/covalent surface of a commerciallyavailable product and on the affinity/covalent surface of Example 5, inembodiments of the disclosure.

FIG. 9 shows a comparison of protein leaching or dissociation responseson an affinity surface, on affinity/covalent surface of a commerciallyavailable product, and on the affinity/covalent surface of Example 5, inembodiments of the disclosure.

FIG. 10 shows a comparison of binding values on an affinity surface, onan affinity/covalent surface of a commercially available product, and onthe affinity/covalent surface of Example 5, in embodiments of thedisclosure.

FIG. 11 shows a comparison of CAII or His-tag CAII immobilization levelson the affinity/covalent surface of Example 6, in embodiments of thedisclosure.

FIG. 12 shows His-tag CAII immobilization levels on theaffinity/covalent surface of Example 7 with or without nickel iontreatment, in embodiments of the disclosure.

FIG. 13 shows four hour attachment human primary hepatocytes on EMA NTAsynthetic surface relative to collagen I.

FIG. 14 shows a comparison of attachment (24 h) and retention (7 d) ofhuman primary hepatocytes on EMA NTA synthetic surface relative tocollagen I.

FIG. 15 shows relative gene expression of three major metabolism genes(CYP450) in human primary hepatocytes cultured on EMA NTA syntheticsurface relative to collagen I.

FIG. 16A-B shows schematic representations of cells on a substrate aloneand in the presence of a sandwich layer which may be, for example,collagen or Matrigel™.

FIG. 17A-C are micrographs of liver tissue (FIG. 17A), liver cells grownon a collagen I coated substrate (FIG. 17B) and liver cells grown on acollagen I coated substrate, and overlaid with a Matrigel™ sandwichlayer (FIG. 17C) fluorescently stained to show MRP2 transporter.

FIGS. 18(A and B) are micrographs of liver cells grown on synthetic EMANTA surface alone (FIG. 18A), and in the presence of a Matrigel™sandwich layer, as illustrated in FIG. 16B.

FIGS. 19(A and B) are micrographs of liver cells grown on synthetic EMANTA surface in the presence of a Matrigel™ sandwich in the presence ofserum (FIG. 19A) and in the absence of serum (FIG. 19B) stained to showMRP2 transporter.

DETAILED DESCRIPTION

Various embodiments of the disclosure will be described in detail withreference to drawings, if any. Reference to various embodiments does notlimit the scope of the invention, which is limited only by the scope ofthe claims attached hereto. Additionally, any examples set forth in thisspecification are not limiting and merely set forth some of the manypossible embodiments for the claimed invention.

Definitions

“Assay,” “assaying,” or like terms refers to an analysis to determine,for example, the presence, absence, quantity, extent, kinetics,dynamics, or type of a biologic's or cell's optical or bioimpedanceresponse upon stimulation with an exogenous stimuli, such as a ligandcandidate compound, a viral particle, a pathogen, a surface or culturecondition, or like entity. Such terms can also include non-biologic ornon-cell responses to stimuli or reactions to stimuli.

“Attach,” “attachment,” “adhere,” “adhered,” “adherent,” “immobilized,”or like terms generally refer to immobilizing or fixing, for example, asurface modifier substance, a compatibilizer, a cell, a ligand candidatecompound, and like entities of the disclosure, to a surface, such as byphysical absorption, chemical bonding, and like processes, orcombinations thereof. Particularly, “cell attachment,” “cell adhesion,”or like terms refer to the interacting or binding of cells to a surface,such as by culturing, or interacting with a cell anchoring material, orlike entity.

“Adherent cells” refers to a cell or a cell line or a cell system, suchas a prokaryotic or eukaryotic cell, that remains associated with,immobilized on, or in certain contact with the outer surface of asubstrate. Such type of cells after culturing can withstand or survivewashing and medium exchanging process, a process that is prerequisite tomany cell-based assays. “Weakly adherent cells” refers to a cell, a cellline, or a cell system, such as a prokaryotic or eukaryotic cell, whichweakly interacts, associates with, or contacts the surface of asubstrate during cell culture. However, these types of cells, forexample, human embryonic kidney (HEK) cells, tend to dissociate easilyfrom the surface of a substrate by physically disturbing approaches,such as washing or medium exchange. “Suspension cells” refer to a cellor a cell line that is preferably cultured in a medium wherein the cellsdo not attach or adhere to the surface of a substrate during theculture. “Cell culture” or “cell culturing” refers to the process bywhich either prokaryotic or eukaryotic cells are grown under controlledconditions. “Cell culture” can also refer to the culturing of cellsderived from multicellular eukaryotes, especially animal cells,including the culturing of complex tissues and organs.

“Cell” or like term refers to a small usually microscopic mass ofprotoplasm bounded externally by a semipermeable membrane, optionallyincluding one or more nuclei and various other organelles, capable aloneor interacting with other like masses of performing all the fundamentalfunctions of life, and forming the smallest structural unit of livingmatter capable of functioning independently including synthetic cellconstructs, cell model systems, and like artificial cellular systems.

“Cell system” or like term refers to a collection of more than one typeof cells (or differentiated forms of a single type of cell), whichinteract with each other, thus performing a biological or physiologicalor pathophysiological function. Such cell system include, for example,an organ, a tissue, a stem cell, a differentiated hepatocyte cell, orlike systems.

“Stimulus,” “therapeutic candidate compound,” “therapeutic candidate,”“prophylactic candidate,” “prophylactic agent,” “ligand candidate,”“ligand,” or like terms refer to a molecule or material, naturallyoccurring or synthetic, which is of interest for its potential tointeract with a cell attached to the biosensor or a pathogen. Atherapeutic or prophylactic candidate can include, for example, achemical compound, a biological molecule, a peptide, a protein, abiological sample, a drug candidate small molecule having, for example,a molecular weight of less than about 1,000 Daltons, a drug candidatebiologic molecule, a drug candidate small molecule-biologic conjugate,and like materials or molecular entity, or combinations thereof, whichcan specifically bind to or interact with at least one of a cellulartarget or a pathogen target such as a protein, DNA, RNA, an ion, alipid, or like structure or component of a live-cell.

“Biosensor” or like terms generally refer to a device for the detectionof an analyte that combines a biological component with aphysicochemical detector component. The biosensor typically consists ofthree parts: a biological component or element (such as a cellulartarget, tissue, microorganism, pathogen, live-cell, or a combinationthereof), a detector element (operating in a physicochemical manner suchas optical, piezoelectric, electrochemical, thermometric, or magnetic),and a transducer associated with both components. In embodiments, anoptical biosensor can comprise an optical transducer for converting amolecular recognition or molecular stimulation event in a cellulartarget, a live-cell, a pathogen, or a combination thereof into aquantifiable signal.

“Include,” “includes,” or like terms refers to including but not limitedto, that is, inclusive and not exclusive.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperature, process time,yields, flow rates, pressures, and like values, and ranges thereof,employed in describing the embodiments of the disclosure, refers tovariation in the numerical quantity that can occur, for example: throughtypical measuring and handling procedures used for making compounds,compositions, concentrates, or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods; and like considerations. The term “about” also encompassesamounts that differ due to aging of a composition or formulation with aparticular initial concentration or mixture, and amounts that differ dueto mixing or processing a composition or formulation with a particularinitial concentration or mixture. The claims appended hereto areintended to include equivalents of these quantities with or without the“about” modifier.

“Optional,” “optionally,” or like terms refer to the subsequentlydescribed event or circumstance can or cannot occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not. For example, the phrase “optionalcomponent” means that the component can or can not be present and thatthe disclosure includes both embodiments including and excluding thecomponent.

“Consisting essentially of” in embodiments refers, for example, to asurface composition, a method of making or using a surface composition,formulation, or composition on the surface of the biosensor, andarticles, devices, or apparatus of the disclosure, and can include thecomponents or steps listed in the claim, plus other components or stepsthat do not materially affect the basic and novel properties of thecompositions, articles, apparatus, and methods of making and use of thedisclosure, such as particular reactants, particular additives oringredients, a particular agent, a particular cell or cell line, aparticular surface modifier or condition, a particular ligand candidate,or like structure, material, or process variable selected. Items thatmay materially affect the basic properties of the components or steps ofthe disclosure or may impart undesirable characteristics to the presentdisclosure include, for example, excessively thick layers of thechelating polymer either as a surface layer, as nanoparticulates, or acombination thereof, such as greater than about 1,000 nm, because of thepenetration depth limits of the evanescent wave, and likeconsiderations.

The indefinite article “a” or “an” and its corresponding definitearticle “the” as used herein means at least one, or one or more, unlessspecified otherwise.

Abbreviations, which are well known to one of ordinary skill in the art,may be used, for example, “h” or “hr” for hour or hours, “g” or “gin”for gram(s), “mL” for milliliters, “rt” or “RT” for room temperature,“nm” for nanometers, and like abbreviations.

“Weight percent,” “wt %,” “percent by weight,” or like terms withreference to, for example, a component, unless specifically stated tothe contrary, refer to the ratio of the weight of the component to thetotal weight of the composition in which the component is included,expressed as a percentage.

Specific and preferred values disclosed for components, ingredients,additives, cell types, antibodies, His-tagged entities, and likeaspects, and ranges thereof, are for illustration only; they do notexclude other defined values or other values within defined ranges. Thecompositions, apparatus, and methods of the disclosure include thosehaving any value or any combination of the values, specific values, morespecific values, and preferred values described herein.

Polymer gels that have been previously described which can immobilizebiomolecules on LID biosensors that are made of, for example, apolysaccharide, such as dextran, or a synthetic polymer, such aspolyacrylic acid, see, for example, U.S. Pat. No. 5,436,161 (assigned toBIAcore), and EP 0,226,470 patent (the “'470 patent”) entitled“Materials and methods for microchemical testing,” J. A. Bosley, et al.,(assigned to Unilever),

Among the numerous types of polymer gels, one frequently used is basedon carboxymethyl dextran as described, for example, in S. Lofas, et al.,“A Novel Hydrogel Matrix on Gold Surfaces in Surface Plasmon ResonanceSensors for Fast and Efficient Covalent Immobilization of Ligands,” J.Chem. Soc., Chem. Commun., 1990, 21, 1526-1528, and the aforementionedU.S. Pat. No. 5,436,161. This carboxymethyl dextran based gel haslimited capture capacity due to its molecular weight (M_(w) 500,000g/mol for the CM5 sensor chip available from BIAcore) which makes theheight or thickness of the final gel unsuitable for very high proteincapture capacity.

A similar approach was described in PCT Publication No. WO 2007/049269(Applicant Bio-Rad). This publication mentions binding layers comprisinga polysaccharide substituted with carboxylic acid groups exhibiting highperformance in the binding of ligand molecules and in the interactionwith analyte molecules. The polysaccharide is modified by reaction withan alanine spacer. This publication mentions that the spacermodification allows more efficient activation of the carboxylic acidgroups of the spacer compared to activation of the carboxylic acidgroups of a known carboxymethylated polysaccharide. This publicationalso mentions that synthetic polymers, like poly(acrylic acid) orpoly(methacrylic acid), exhibit much more efficient activation andsubsequent immobilization. However, the ligand molecules exhibited lowactivity perhaps due to lower “biocompatibility” of these polymers (seep. 13, lines 5-9).

The LID method is based on the local change of refractive index inducedby the adsorption of a ligand onto an immobilized target such as areceptor(s).

The issue of low target immobilization on a biosensor and low specificligand binding activity on a biosensor can be overcome by theselectively chemically and biologically functionalized surfaces of thedisclosure having high biochemical target immobilization on a biosensorand high specific ligand binding activity with the surface boundbiochemical target.

The surface disposition of the biosensor article of the disclosureenhances the resulting signal by increasing the immobilized receptordensity, i.e., the number of the receptors immobilized on the biosensorwhich in turn provides increased capture capacity for targeted ligands,and by providing increased activity or availability of the immobilizedreceptor.

In embodiments, the surface chemistry of the disclosure may becompatible with Dual Polarized Intereferometry (DPI), which is anothertype of LID sensor, or surface plasmon resonance (SPR) type sensors.

In embodiments, the disclosure relates to a method for efficientlyimmobilizing biomolecules, such as proteins, for example, on a substrateor sensor surface. The disclosure is particularly useful in the field ofbiosensors for label independent detection (LID). The disclosure relatesmore particularly to surface chemistry of LID biosensors and a method ofpreparation.

The disclosure more specifically concerns a sequential method forproviding chemically modified surfaces that are capable of immobilizing,for example, receptors (e.g. proteins) by affinity capture at higherdensity and higher stability than previously reported. The LID biosensormade according to the disclosure has a higher sensitivity for thedetection of bio-molecular recognition events compared to known methods.Alternatively or additionally, the immobilization surface does notrequire any pre-activation, which is otherwise generally time consumingand a source of variability. The disclosure is particularly well suitedfor LID biosensors, for example, an Epic® system (Corning®Incorporated), those based on surface plasmon resonance (SPR), DualPolarized Interferometry, and like methodologies.

In the analysis of the biomolecular recognition events with LID, atleast one biomolecule must be immobilized on the substrate as close aspossible to the wave guide surface and a second molecule partner orcomplement, which recognizes or is recognized by the immobilizedbiomolecule, and reacts or binds to the immobilized molecule. Bindinglocally modifies the refractive index due to the local mass increase andis detected as a wavelength shift or SPR signal.

For LID techniques based on such a local change of refractive indexinduced by the adsorption of the ligand onto the immobilized receptors,proper surface chemistry can enhance the signal by increasing the numberof the receptors immobilized so as to capture a greater amount ofligands. Moreover, the surface chemistry must retain the receptor firmlyattached on the sensor surface. Indeed, desorption of the receptor leadsto an unacceptable huge variation of the wavelength shift or the SPRsignal which may totally hide the binding of the small molecule, such asa new drug entity. To prevent such receptor desorption, covalentattachment of the receptor on the sensor is commonly used. But in somecases, covalent immobilization can lead to partial or complete loss ofprotein activity, due to random orientation, structural deformation, andlike considerations.

To achieve a high binding response, it is desirable to have a high levelof immobilized biomolecules, and equally desirable to have immobilizedbiomolecules available for the binding event. This means thatbiomolecules must be in a native or active conformation, andwell-oriented on the sensor surface to prevent, for example, sterichindrance effects which generally lead to a reduced binding response.

To obtain such biomolecule availability and good orientation,immobilization through affinity capture is generally preferred tocovalent attachment. Such affinity capture methods are, for example,based on biotinylated molecules captured by streptavidin or avidinpreviously attached on the surface, or histidine-tagged moleculescaptured by a metal ion previously immobilized on the surface. Both ofthese methods require the addition of a tag to the biomolecule for theimmobilization step but can provide excellent availability and goodorientation of the immobilized biomolecules. However, for immobilizedhistidine tagged molecules, desorption is usually observed and can hidebinding of small molecules.

The disclosure provides a sequential method for providing biosensorsurfaces capable of immobilizing receptors (e.g., proteins) by affinitycapture at higher density and higher stability than previously reported,followed by a chemical treatment step that leads to covalent attachmentof the receptor(s) on the substrate.

Metal chelate affinity chromatography, reported by Porath (J. Porath, etal., “Metal Chelate Affinity Chromatography, A New Approach to ProteinFractionation,” Nature, 1975, Vol. 258, pg. 598), is one known techniquefor fractionation of proteins by chromatography. This technique allowsone to selective capture proteins onto a stationary phase previouslyfunctionalized with a metal ion/iminodiacetic acid complex.

Hochuli has described a new metal chelate resin made by graftingcarboxymethyl lysine (CML) groups, for example, onto a carrier matrix.The nitrilotriacetic acid (NTA) groups grafted to the matrix provide anefficient chelate resin which exhibits a stronger Ni²⁺ attachmentcompared to the previously reported iminodiacetic acid based resin (seeU.S. Pat. No. 4,887,830, Hochuli, et al., “Metal Chelate Resins,”European Patent No. EP 0 253 303B1, Hochuli, et al., “NeueMetallchelatharze,” and E. Hochuli, et al., J. Chromatogr., 1987, Vol.411, pgs. 177-184). This nitrilotriacetic acid group can selectivelybind proteins and peptides containing neighboring histidine residues.The method of preparing the nitrilotriacetic compound is described in,e.g., U.S. Pat. No. 4,877,830.

Due to this significant improvement of the metal/chelate complexstability, nitrilotriacetic acid (NTA)/histidine-tag (HT) technology hasbecome a powerful tool for isolation and purification of recombinantproteins and enzymes modified at their N- or C-termini with histidineresidues (K. Terpe, et al., Microbiol. Biotechnol., 2003, Vol. 60, pgs.523-533).

Despite that the NTA/metal/HT interaction is suitable for proteinpurification, applications requiring longer-term stability such asbiosensors, surface coatings, and binding studies are compromised byproblems related to metal leaching and protein dissociation (see, e.g.,W. Jiang, et al., “Protein Selectivity with Immobilized Metal Ion-tacnSorbents: Chromatography Studies with Human Serum Proteins and SeveralOther Globular Proteins,” Anal. Biochem., 1998, Vol. 255, pgs. 47-58,and Mateo, et al., Biotechnol. Bioeng., 2001, Vol. 49, pgs. 313-334).The immobilization of the His-tagged protein to the Ni²⁺/NTA surfaceneeds to be stable especially if the kinetics of interactions betweenimmobilized proteins and a further partner, such as a drug are, forexample, low. It is known that the stability of the metal ion to NTA ishigh due to the very high affinity of the metal to the NTA group asdemonstrated by Hochuli.

Unfortunately, the histidine tag (His-tag) binds with only low affinityon the metal ion. Nieba estimate the dissociation constant to about 10⁻⁶M at neutral pH (see L. Nieba, et al., “Bioacore Analysis ofHistidine—Tagged Proteins Using a Chelating NTA Sensor Chip,” Anal.Biochem., 1997, Vol. 252, pgs. 217-228). While this is acceptable forprotein purification by immobilized metal ion-affinity chromatography(IMAC), it is less well suited for immobilization on sensors andparticularly LID sensors. For these reasons and in spite of its numerousadvantages, the NTA capture approach is rarely used for ligand screeningusing LID, because it is known (see e.g., Gershon, infra.) to causesubstantial error in binding responses due to the inevitable proteindissociation.

The present disclosure, in embodiments, provides surface chemistry basedon NTA capture but provides further improvements, which for exampleprevents protein dissociation.

However, despite the risk of protein dissociation, Whitesides, et al.,(see, e.g., G. B. Sigal, et al., “A Self-Assembled Monolayer for theBinding and Study of Histidine-Tagged Proteins by Surface PlasmonResonance,” Anal. Chem., 1996, Vol. 68, pgs. 490-497, and U.S. Pat. No.5,620,850, Whitesides, et al., “Molecular Recognition at SurfacesDerivatized with Self-Assembled Monolayers”) described the use of NTAchemistry on SPR sensors for protein capture and analysis ofprotein/protein interactions. Whiteside, et al., mentioned that thismethod offers numerous advantages over methods that historically havebeen used for immobilizing proteins on gold surfaces, including covalentattachment to a carboxylated dextran layer. Indeed, this surfacechemistry allows immobilization of protein containing His-tag with ahigher percentage of protein recognizable by antibodies and othersproteins than can be achieved by covalent modification.

Unfortunately, Whiteside's paper again shows that the protein is morerecognizable when immobilized by means of NTA capture chemistry due tothe well controlled protein orientation. However, the amount of proteinimmobilized remains too low for LID application. Whitesides reportedthat its self-assembled monolayer (SAM) functionalized with the NTAyields to about 1 ng/mm² of proteins captured (about 1,000 RUimmobilization response). Although such a low quantity of immobilizedprotein is acceptable for large molecule interaction assays such asprotein/antibody or protein/protein assays, it is unsuitable for smallmolecule/protein assays such as drug/protein assays. The poorimmobilization performances of NTA chemistry prepared according to knownprocesses, makes the NTA capture chemistry unsuitable for drug discoveryusing LID sensors.

Additionally, the problem of low immobilization capacity of an NTAmonolayer has been studied by Gershon (P. D. Gershon, et al., “StableChelating Linkage for Reversible Immobilization of Oligohistidine TaggedProteins in the Biacore Surface Plasmon Resonance Detector,” Journal ofImmunological Methods, 1995, Vol. 183, pgs. 65-76), who proposed an SPRchip coated with NTA modified dextran hydrogel. This chemistry is nowcommercially available from BIAcore under the trade name NTA-chip. Thedextran matrix provides a 3D hydrogel (typically 100 nm thick) whichallows capturing significantly much more protein than observed withsurface chemistry based on an NTA monolayer (SAM) described above.Although this NTA sensor chip is particularly useful for large moleculeinteraction studies, it remains unsuitable for small moleculeinteractions due to the limited amount of immobilized proteins and tohigh protein dissociation.

To overcome the drawback of protein leaching, another strategy hasdescribed how to combine affinity capture and covalent coupling toensure good protein stability and prevent protein dissociation, see WO2004/046724;; U.S. Patent Publication No. US2006/0014232, to Inagawa, etal., “Immobilization Method”; and F. S. Willard, et al., “CovalentImmobilization of Histidine-Tagged Proteins for Surface PlasmonResonance,” Anal. Biochem., 2006, Vol. 353, pgs. 147-149. Unfortunately,this method suffers from an important drawback: as covalent coupling andaffinity capture are realized at the same time, protein attachments areperformed in an uncontrolled manner by reaction between reactive groupsof the surface and reactive groups from the proteins without anyspecificity. This is particularly unsuitable because the main reason toselect capture of protein by affinity is that the protein is attached tothe sensor surface by means of only the tag having a well definedposition and not by a non-specific chemical reaction. Another strategywas applied using the NTA-chip. After immobilization of proteins on theNTA-chip, an EDC/NHS treatment was performed on immobilized biomoleculesto create covalent bonds between the substrate and the biomolecules (seeM. A. Wear, et al., “A Surface Plasmon Resonance-Based Assay for SmallMolecule Inhibitors of Human Cyclophilin A,” Anal. Biochem., 2005, Vol.345, pgs. 214-226). Unfortunately, even if no dissociation ofbiomolecules from the substrate was observed after the EDC/NHStreatment, protein leaching appears to be important during theactivation step. Coupled with the inherently poor immobilizationcapacity of the chemistry of known methods, protein leaching or lossfrom the surface further contributes to a decrease in the amount ofimmobilized protein on the substrate. Finally, the low capacity ofbiomolecules captured appears to be a primary drawback that preventsthis chemistry from being suitable for small molecule recognitionevents.

Attempts to attach NTA chelating groups on metal nanoparticles such asgold-nanoparticles (see Abad, et al., “Functionalization of ThioaceticAcid-Capped Nanoparticles for Specific Immobilization of HistidineTagged Proteins,” J. Am. Chem. Soc., 2005, Vol. 127, pgs. 5689-5694) ormagnetic-nanoparticles (see Xu, et al., “Nitrilotriacetic Acid-ModifiedMagnetic Nanoparticles as a General Agent to Bind Histidine taggedProteins,” J. Am. Chem. Soc., 2004, Vol. 126, pgs. 3392-3393) have beendescribed but they do not mention how to enhance efficiency of LIDsensors with polymer only based nanoparticles.

In embodiments, the present disclosure provides affinity based surfacechemistry that has, for example, a very high immobilization capacity,which is compatible with label free detection, which provides a highstability NTA-Ni-histidine complex, and which prevents substantially anyprotein dissociation using a post-immobilization covalent attachmentreaction prior to ligand detection.

In embodiments, the disclosure provides a biosensor article comprising:

a substrate having nanoparticles (NP) on the substrate surface, thenanoparticle comprises:

-   -   a polymer of formula (I)

having at least one of: a metal-ion chelating group (x), an ionizablegroup (y), and a surface substantive group (z),where

R is hydrogen or a substituted or unsubstituted, linear or branched,monovalent hydrocarbyl moiety having from 1 to 6 carbon atoms;

R′ is a substituted or unsubstituted, linear or branched, divalenthydrocarbyl moiety resulting from copolymerization of an unsaturatedmonomer having from 2 to 10 carbon atoms with, for example, maleicanhydride;

R″ is a substituted or unsubstituted, linear or branched, divalenthydrocarbyl moiety having from 1 to 10 carbon atoms;

S comprises at least one point of attachment to a substrate, and can be,for example, a substrate surface group or modified surface group thatcan covalently bond to the polymer via the X substituent;

W comprises at least one metal-ion chelate groups, and is an incipientbinding site for a biomolecule having at least one tag, that is W canbe, for example, at least one bi-dentate group, such as a metal-ionchelating group in the absence of a metal-ion, and can be, for example,an active binding site for a biomolecule having at least one tag in thepresence of a chelated metal ion.

X can be, for example, —NH—, —NR—, or O, and like divalent groups;

the mole ratio of x:(y+z) groups can be, for example, from about 2:8 toabout 8:2; and the nanoparticles can have, for example, a diameter offrom about 10 to about 100 nanometers.

In embodiments, the disclosure provides a cell culture articlecomprising:

a substrate having nanoparticles (NP) on the substrate surface, thenanoparticle comprises:

-   -   a polymer of formula (I)

having at least one of: a metal-ion chelating group (x), an ionizablegroup (y), and a surface substantive group (z),where

R is hydrogen or a substituted or unsubstituted, linear or branched,monovalent hydrocarbyl moiety having from 1 to 6 carbon atoms;

R′ is a substituted or unsubstituted, linear or branched, divalenthydrocarbyl moiety resulting from copolymerization of an unsaturatedmonomer having from 2 to 18 carbon atoms with maleic anhydride;

R″ is a substituted or unsubstituted, linear or branched, divalenthydrocarbyl moiety having from 1 to 20 carbon atoms;

S comprises at least one point of attachment to the substrate;

W comprises at least one bi-dentate group;

X is an —NH—, —NR—, or O;

the mole ratio of x:(y+z) groups is from about 2:8 to about 8:2; and thenanoparticles have a diameter of from about 10 to about 100 nanometers.

In embodiments, R is a hydroxy substituted, monovalent hydrocarbylmoiety having from 2 to 6 carbon atoms.

In embodiments, the mole ratio x:(y+z) is about 1:1.

In embodiments, S comprises at least one of: a metal oxide, a mixedmetal oxide, a polymer, a composite, or a combination thereof; a surfacemodified substrate; or a combination thereof.

In embodiments, R is a hydroxy substituted alkyl having from 2 to 4carbon atoms; R′ is a divalent hydrocarbyl moiety having from 2 to 10carbon atoms; R″ is a substituted or unsubstituted, divalent hydrocarbylmoiety having from 3 to 6 carbon atoms; S is an aminosiloxane treatedglass or plastic substrate; W comprises at least one iminodiacetic acid,nitrilotriacetic acid, triazacyclononane, aminoethylethanolamine,triethylenetetramine, 2-hydroxypropane-1,2,3-tricarboxylate, or amixture thereof; X is —NH—; the mole ratio x:(y+z) is from about 2:1 toabout 1:2; and the nanoparticles have a diameter of from about 10 toabout 100 nanometers.

In embodiments, R is a hydroxy substituted alkyl having from 2 to 4carbon atoms; R′ is a divalent hydrocarbyl moiety having from 2 to 10carbon atoms;

R″ is a substituted or unsubstituted, divalent hydrocarbyl moiety havingfrom 3 to 6 carbon atoms; S is a plastic substrate; W comprisesnitrilotriacetic acid; X is —NH—;

the mole ratio x:(y+z) is from about 2:1 to about 1:2; and thenanoparticles have a diameter of from about 10 to about 100 nanometers.

In embodiments, the disclosure provides a method of culturing cellscomprising:

providing a cell culture substrate as described; providing cells to thecell culture substrate; incubating the cells in a suitable media on thecell culture substrate. In embodiments, the disclosure providesproviding a sandwich layer to the cells on the cell culture substrate inthe presence of or absence of serum. In embodiments, the cells arehepatocytes.

In embodiments, the R group in (y) can be, for example, a hydroxysubstituted, monovalent hydrocarbyl moiety having from 2 to 6 carbonatoms, for example, the product of the polymer, for example havingresidual anhydride groups, and ethanol amine. In embodiments, R can be,for example, hydrogen, or a hydroxy substituted, linear or branchedalkyl having from 2 to 4 carbon atoms, such as ethylene, propylene,butylene, and like substituted hydrocarbyl moieties.

In embodiments, the R′ can be, for example, a divalent hydrocarbylmoiety having from 2 to 10 carbon atoms. R′ can be, for example,ethylene, propylene, isobutylene, styrene, and like hydrocarbylmoieties, see for example, PCT/US2006/047885, at page 12.

In embodiments, the R″ can be, for example, a substituted orunsubstituted, divalent hydrocarbyl moiety having from 3 to 6 carbonatoms.

In embodiments, the S can be, for example, at least one of: a metaloxide, a mixed metal oxide, a polymer, a composite, or a combinationthereof, for example, Nb₂O₅, SiO₂, Nb₂O₅/SiO₂, cyclic olefin copolymer,and like points of attachment to a substrate. S can be, for example, anaminosiloxane treated glass or plastic substrate. The S can additionallyor alternatively be, for example, a surface coating modified substrate,such as with known silanes GAPS, APS, MOPS, and like modifiers, or acombination thereof.

In embodiments, the W chelating group can be, for example, at least oneiminodiacetic acid (IDA), nitrilotriacetic acid (NTA), triazacyclononane(TACN), aminoethylethanolamine, triethylenetetramine,2-hydroxypropane-1,2,3-tricarboxylate (citrate), and like bis-, tris-,and multi-dentate chelating substituents, derivatives thereof, or acombination thereof. The metal-chelate group can be mono-NTA, bis-NTA ortris-NTA, tetrakis-NTA, poly-NTA, and like groups, or a combinationthereof. The ionizable groups can be, for example, carboxylic acidgroups, and like groups, or a combination thereof.

In embodiments, the X can be, for example, —NH— (an amic-acid), —O— (ahemi-ester), and like groups, or a combination thereof. In embodiments,a preferred X is —NH—.

In embodiments, the mole ratio x:(y+z) can be, for example, from about2:8 to about 8:2; from about 2:1 to about 1:2; and from about 1 to about1, including all intermediate values and ranges. The mole ratio ofx:(y+z) can preferably be, for example, about 1:1, such as found in theexemplary EMA050NTA material disclosed herein.

In embodiments, the nanoparticles (NP) on the substrate surface can be,for example, a layer of nanoparticles, polymer film, or a combination ofnanoparticles and polymer film. The polymeric nanoparticle layer can be,for example, a coat or coating of nanoparticles, by analogy for examplewith “a coating of dust or snow (particles)” and which layer or coatingcan have, for example, complete or incomplete substrate surfacecoverage. The nanoparticles can have a particle diameter thickness of,for example, from about 10 to about 1,000 nanometers, from about 20 toabout 1,000 nanometers, from about 20 to about 500 nanometers, fromabout 20 to about 300 nanometers, from about 20 to about 200 nanometers,from about 20 to about 150 nanometers, from about 20 to about 100nanometers, from about 20 to about 75 nanometers, and like particlediameter thickness, including all intermediate ranges and values. Inembodiments, the nanoparticles can have a diameter of, for example, fromabout 10 to about 100 nanometers, from about 10 to about 60 nanometersand from about 20 to about 40 nanometers, including all intermediateranges and values.

A brief summary of one illustrative preparative procedure for making abiosensor article includes, for example, coating a suitable substrate,such as an insert or glass slide with a tie-layer or conversion coating,such as obtained by treatment with APS. Next, the tie-layer treatedsubstrate can be, for example, dip-coated in a solution or dispersion ofthe polymer, such as an EMA050NTA solution at a concentration of 1 mg/mLin DMSO:IPA=50/50=v:v, over about 10 min. The rinsed and dried polymercoated substrate is then treated with ethanolamine or like agent, eitherneat or in a suitable solution such as a borate buffer, for about 30min. The metal ion can be complexed with the polymer by, for example,the addition of a suitable metal salt solution, such as 40 mM NiSO₄solution, and stirring for about 30 min.

In embodiments, the polymer can be, for example, a preformed derivatizedproduct of a maleic anhydride (MA) polymer or ethylene-maleic anhydride(EMA) copolymer having a portion of the backbone derivatized with aspacer (R″), at least one bi-dentate group such as a metal-ion chelator(W), and optionally a metal-ion (M^(n+)). In embodiments, the polymercan alternatively be prepared, for example, by contacting thenanoparticle decorated substrate surface and a compound having a spacerand a metal chelator (e.g., carboxymethyl lysine; CML also known asNTA), and then contacting the nanoparticle decorated substrate havingthe attached spacer and a bi-dentate metal-ion chelator, and a metal-ionsolution to form nanoparticles having at least some chelated metal ion.

FIG. 1B shows an exemplary structure of the polymer of formula (I)associated with a surface (S). Although not limited by theory, it isbelieved that nanoparticles of the polymer of formula (I) can reversiblyrelax or unfold in certain solutions, such as buffer media, to providean extended structure more akin to sea-weed attached to the bottom oflake by a single or very few points of attachment. The extendedstructure can provide a greater number of points where the metal ion maycomplex and thus where a tagged biomolecule or like entity can complex.

In embodiments, the disclosure provides a method for using the disclosedbiosensor article comprising:

contacting the substrate having the abovementioned metal-ion complexednanoparticles (NP) and a His-tagged entity target, such as a smallmolecule, Ab, protein, cell, and like entities, having at least oneHis-tag or label, for example, His-tagged carbonic anhydrase, toimmobilize the His-tagged entity, and

contacting the nanoparticles having the immobilized His-tagged entityand a stabilizing agent to form a nanoparticle decorated substratehaving a stabilized His-tagged entity.

The stabilizing agent can be, for example, NHS/EDC, and like reagents ortreatments. The stabilized intermediate product resulting from thecontacting with the stabilizing agent need not be isolated. If desired,stabilized intermediate product can be used directly in covalent ligandcapture and sensing. The method of use can further include contactingthe nanoparticle decorated substrate having the stabilized His-taggedentity, with a ligand to form a nanoparticle decorated substrate havinga His-tagged entity having a bound ligand, i.e., a ligand for theHis-tagged entity or His-tag conjugate-former, e.g., small molecule, Ab,protein, cell, and like ligands. The His-tag target affinity capture,the stabilization step, and the covalent ligand capture and sensing areschematically illustrated in FIG. 1C.

In embodiments, contacting the nanoparticle decorated substrate with aHis-tagged entity, i.e., immobilization, can be accomplished at, forexample, a pH of from about 3 to about 9. The contacting of thenanoparticle decorated substrate with a His-tagged entity can beaccomplished at, for example, a pH above the pI of the His-taggedentity.

In embodiments, the His-tagged entity immobilization can be, forexample, greater than about 1,500 pm and the loss of immobilizedHis-tagged entity can be, for example, less than about 0.1 wt % of thetotal originally immobilized His-tagged entity, which increasedimmobilization and stability properties taken together provide abiosensor having a biosensor binding response greater than about 10picometers (pm). The biosensor can be, for example, at least one of asurface plasmon resonance biosensor, a waveguide resonant gratingbiosensor, an impedance biosensor, a mass spectrometry biosensor, andlike devices, or a combination thereof.

In embodiments, the disclosure provides a method for performing an assayof a ligand, the method comprising:

contacting the ligand and a biosensor article as disclosed herein, suchthat if the ligand binds to the His-tagged entity, then: detecting theligand-induced response of the biosensor.

In embodiments, the His-tagged entity can be, for example, at least oneof: a natural or synthetic oligonucleotide, a natural or syntheticnucleic acid (DNA or RNA), a natural peptide, a natural or syntheticpeptide optionally comprising one or more modified or blocked aminoacids, an antibody, a hapten, a biological ligand, a protein membrane, alipid membrane, a protein, a small molecule having a molecular weight ofless than about 500 Daltons, a cell, or a combination thereof, or aconjugate thereof, where the His-tagged entity has at least one His-tagor His-label, and preferably more than one His-tag or His-label, such asfrom about two to about six, including intermediate values and ranges.

In embodiments, the ligand can be, for example, at least one of: astimulus, a therapeutic candidate, a prophylactic candidate, aprophylactic agent, a chemical compound, a biological molecule, apeptide, a protein, a biological sample, a small molecule having amolecular weight of less than about 500 Daltons, a biologic drugmolecule candidate, a drug candidate small molecule-biologic conjugate,a pathogen, a cell, or combinations thereof. While the ligand mayinclude a His-tag or His-label, the ligand need not include a His-tag orHis-label to be operational in binding or biosensing embodiments of thedisclosure.

In embodiments, the disclosure provides a method for using the biosensorarticle as disclosed herein, including, for example, contacting thenanoparticle decorated substrate having the His-tagged entity and astabilizing agent to form a nanoparticle decorated substrate having astabilized His-tagged entity. The method can further include contactingthe resulting nanoparticle decorated substrate having a stabilizedHis-tagged entity and a ligand to form a nanoparticle decoratedsubstrate having the His-tagged entity now having a bound ligand.

In embodiments, the disclosure provides an article prepared by the abovemethod for use in a biosensor or a cell culture.

In embodiments, a polymer of the formula (I) can be, for example,Formula I(a):

In embodiments, a polymer of the formula (I) can be, for example, of theformula I(b):

In embodiments, a polymer of the formula (I) when complexed with ametal-ion such as Ni²⁺, can be, for example, of the formula I(c):

When a LID biosensor such as grating resonant sensors or SPR-sensors areused, the evanescent field wave can only probe about the first 100 toabout 200 nanometers from the surface making a micrometer thick gelunsuitable. Thus, biomolecules that have been captured in the gel beyondabout 200 nm from the sensor surface are effectively invisible to theevanescent field wave, and any biomolecular recognition event occurringbeyond about 200 nm is not detected. This situation yields unacceptablyhigh biomolecule consumption, which further limits it's applicabilityfor high throughput system (HTS) applications, such as those involvingprecious protein.

In embodiments, the disclosure provides a surface chemistry, based onsynthetic polymers, which can be easily attached to a LID sensorsurface, has very high immobilization capacity, provides goodavailability and activity of the immobilized biomolecule, and iscompatible with label-free detection methodologies. The method of makingis easy to implement, and is compatible with manufacture scale-up, e.g.,does not require long polymerization times nor long washing times.

In embodiments, the disclosure provides a surface chemistry, based onsynthetic polymers, which can be attached to a substrate suitable forcell culture applications. In embodiments, the surface chemistry is ananoparticle layer on the surface of a substrate. In embodiments, asubstrate suitable for cell culture application may be, for example,glass, metal, ceramic, or polymeric material. Suitable polymericmaterial may be any substrate known in the art and may include, forexample, polyacrylates, polymethylacrylates, polycarbonates,polystyrenes, polysulphones, polyhydroxy acids, polyanhydrides,polyorthoesters, polypropylenes, polyphosphazenes, polyphosphates,polyesters, cyclic polyolefin copolymers or mixtures thereof, which maybe surface mofidied (for example plasma treated) or untreated. Inembodiments, suitable cell culture articles include microwell plates,slides, flasks, cell stack or multi-layer cell culture articles such asCorning's HyperFlask™ or HyperStack™ products. In embodiments, thenanoparticle layer on the surface of the substrate is a polymer ofFormula I. In embodiments, the nanoparticle layer on the surface of thesubstrate is Formula I(a) or Formula I(b) where there is no metalchelated to the metal chelating group (x). In embodiments, thenanoparticle layer for cell culture applications is the nanoparticlelayer illustrated in FIG. 1B. In embodiments, the cell culture articleis suitable for culturing cells including primary cells, for example,human primary hepatocytes, cell lines including, for example, HepG2/C3A(ATCC #CRL-10741, 3-20 passages), HepG2 (ATCC #HB-8065, 3-20 passages),Fa2N-4 (commercially available from XenoTech, passages 32-34), or thelike. These cells may be cultured in any suitable media including, forexample, media commercially available from XenoTech, Invitrogen or thelike.

Experiments comparing four hour attachment of human primary hepatocyteson EMA NTA synthetic surface (as shown in FIG. 1B) relative to collagenI show that the synthetic surface, EMA NTA supports attachment of humanprimary hepatocytes similar to collagen I in serum culture conditionsfor four hours. The expression of bile canaliculi and apical domain arephysiologically relevant markers for hepatocytes in culture. MRP2transporter is a marker of the bile canaliculi structures at the apicaldomain between hepatocytes and indicates the degree of restoration ofcell membrane polarity of cells in culture. Extended and networked bilecanaliculi structures indicate good (in vivo like) restoration of cellpolarity and a more in vivo-like cell morphology. EMA NTA surface showssimilar performance to collagen; without the Matrigel™ overlay(sandwich), hepatocyte polarity (in vivo like morphology) is notrestored, and with the Matrigel™ overlay (sandwich), hepatocyte polarity(in vivo like morphology) is restored to a relatively greater degreethan cells growing on other experimental cell culture surfaces (data notshown). These extended and networked bile canaliculi structures can beseen in FIGS. 17 (FIG. 17 illustrates control surfaces) 18 and 19.

Experiments testing the ability of the EMA NTA surface to support theculture of cells in the presence and absence of serum show that cellsare successfully cultured on the EMA NTA surface in the presence and inthe absence of serum. For example, FIG. 15 shows that gene expression isdistributed more homogeneously across the EMA NTA surfaces in thepresence of serum. FIG. 19 shows that there may be more crowding andclustering of cells in the serum free condition (FIG. 19B) than instandard culture conditions with serum (see FIG. 17 and see also FIG.19A on the EMA NTA surface in the presence of serum). Bile canaliculistructures appear to be further extended and more interconnected in theregions where cells are clustered under serum free culture conditions.This may explain the slight increase in function (gene expression)observed in the serum free conditions (see FIG. 15).

By using the described preparative method, a very reproduciblenanoparticle layer on the surface of the substrate can be obtained whichis fully compatible with commercially available LID systems such as theEpic® system (Corning Incorporated) and with the preparation of cellculture articles.

The nanoparticle layer on the surface of the substrate provides enhancedcapture of the biomolecules as demonstrated in the working examples. Thenanoparticle layer on the surface of the substrate can be, for example,from about 10 to about 100 nm, as measured by, for example, SEM or AFMmethods.

Because a very high immobilization level of protein can be obtained anda high activity of the protein established, the coated sensor article ofthe disclosure can be particularly suitable for detecting binding eventsoccurring between proteins and very low molecular weight molecules,e.g., small molecules.

Alternative or additional reactive comonomers can be selected whichcomprise or can be converted after in-situ polymerization to aldehyde,azide, epoxy, isocyanate, isothiocyanate, sulfonyl chloride, carbonate,maleimide, acyl imidazole, aziridine, imidazole carbamate, succinimidylester and succinmidyl carbonate, hydrazone, iminodiacetic acid, nitrilotriacetic acid, triazacyclononane, thiol, substituted disulfide groupsuch as pyridine disulfide, and like groups. A panel of reactive groupsthat can be used with the present disclosure is disclosed, e.g., in“Bioconjugate Techniques”, Greg T. Hermanson, Academic Press, 1996. Whenthe reactive group is likely to interfere with the polymerization of themonomers mixture, the reactive group can be, for example, introducedafter the in-situ polymerization or can be protected using a protectinggroup methods.

The present disclosure provides a process for preparing LID sensorshaving unexpectedly high protein immobilization capacity whilesignificantly improving the protein activity which dramatically enhancesthe assay sensitivity, as illustrated and demonstrated herein. Theimmobilization and binding responses of the biosensor articles of thedisclosure out-perform other known surface chemistries. For example, theEpic® binding response for a furosemide/carbonic anhydrase assay asdisclosed herein, can be, for example, at least about 2 to about 10times higher than the those obtained with a sensor made according to themethod described in WO 2007/078873.

In embodiments, the sensor of the disclosure is compatible with highthroughput screening (HTS) of drug or other small molecules due to thehigh binding response provided. Even very low molecular weightcompounds, such as fragments having a molecular weight of less thanabout 500 Daltons, can be also screened using the sensor due to the highbinding response.

In embodiments, the disclosure permits preparation of sensors forlabel-free detection using a low protein concentration which suggeststhat the cost per analysis can be substantially reduced compared toother LID techniques.

In embodiments, the disclosure provides a surface that is suitable forthe attachment, growth, and assay of many types of cells, includingstrongly adherent cells such as Chinese hamster ovary (CHO) cells andhuman epithelial carcinoma A431 cells, intermediate adherent cells suchas RMS13 cells, and weakly adherent cells such as human embryonic kidney(HEK) cells, or primary cells.

The disclosure provides methods to modify the surface of a biosensor sothat the surface of these biosensors is compatible with and amenable tocell culture and subsequent cell assays. The disclosed method issuitable for oxidized metal thin film surfaces such as the ones used inresonant waveguide grating biosensors, or an un-patterned gold surface,such as those used in surface plasmon resonance (SPR), or a patternedgold surface, such as those used in electrical bioimpedance-basedbiosensors.

The disclosure may suitably comprise, consist of, or consist essentiallyof: a cell culture article as defined herein; a method for preparing thecell culture article as defined herein; and a method for performing anassay of a ligand as defined herein. In embodiments, the disclosureprovides a cell culture article comprising: a substrate; an optionaltie-layer attached to at least the substrate; and a bio-compatible layerof the disclosed polymer attached to the optional tie layer, to thesubstrate, or both.

The substrate can comprise, for example, a plastic, a polymeric orco-polymeric substance, a ceramic, a glass, a metal, a crystallinematerial, a noble or semi-noble metal, a metallic or non-metallic oxide,a transition metal, or a combination thereof In embodiments, thetie-layer can be obtained from a compound comprising one or morereactive functional groups comprising, for example, an amino group, athiol group, a hydroxyl group, a carboxyl group, an acrylic acid, anorganic or inorganic acid, an ester, an anhydride, an aldehyde, anepoxide, and like groups, and salts thereof, or a combination thereof.The choice of materials for forming the tie-layer can depend on thenature of the substrate. For example, Slane can be an excellenttie-layer in conjunction with an oxidized inorganic substrate such asglass, SiO_(x)-presenting substrate, TiO₂, Nb₂O₅, Ta₂O₅, HfO₂, andmixtures thereof, or like substrate. Alternatively or additionally, theaforementioned inorganic substrates can be combined with a SiO_(x)overlay. A thiol compound can be an excellent tie-layer when a goldsubstrate is selected. A positively charged polymer such as poly-lysinecan be an excellent tie-layer when a polymeric substrate is used.

In embodiments, the tie layer can be obtained from, for example, astraight or branched-chain aminosilane, aminoalkoxysilane,aminoalkylsilane, aminoarylsilane, aminoaryloxysilane, or like silanesor salt thereof, and combinations thereof. Specific examples ofcompounds that can be used to form the tie layer include, for example,3-aminopropyl trimethoxysilane, N-(beta-aminoethyl)-3-aminopropyltrimethoxysilane, N-(beta-aminoethyl)-3-aminopropyl triethoxysilane,N′-(beta-aminoethyl)-3-aminopropyl methoxysilane,aminopropylsilsesquixoane, or like compounds, and combinations thereof.In a preferred embodiment, the tie layer can be, for example,aminopropylsilsesquioxane, the polymer can be, for example,poly(alkylene-co-maleic anhydride), and the substrate can be, forexample, a microplate or a microscope slide. In embodiments, the tielayer can be, for example, poly-lysine, polyethyleneimine, and likesubstantive polymers, or combinations thereof. Pludemann mentionssurface modifiers, a silicone elastomer or like rubber applications,such as articles or devices, and like applications, in Silane CouplingAgents, (1982). For additional definitions, descriptions, and methods ofsilica materials and related metal oxide materials, see for example, R.K. Iler, The Chemistry of Silica, Wiley-Interscience, 1979.

In embodiments, the bio-compatible nanoparticulate polymer layer canhave a thickness, for example, of from about 10 Å to about 2,000 Å, fromabout 10 Å to about 1,500 Å, from about 10 Å to about 1,250 Å, fromabout 10 Å to about 1,000 Å, and from about 10 Å to about 100 Å. Inembodiments, the polymer layer that forms the bio-compatible layer, ifinitially continuous, can be ruptured or disrupted during an extended ora vigorous oxidation process to provide bio-compatible layers thatincludes gaps or regions with little or no coverage of the underlyingtie-layer or the substrate surface, that is a discontinuous layer orfilm of the bio-compatible nanoparticulate polymer layer can result.Similarly, a discontinuous layer or film of the bio-compatiblenanoparticulate polymer layer can result from less extensive or lessvigorous oxidation of an initially discontinuous polymer layer.Accordingly, the bio-compatible nanoparticulate polymer layer havingruptured areas or a discontinuous layer can have layer thicknesses of,for example, from about 10 to about 200 Å. In embodiments, thenanoparticulate polymer can have a thickness of from about 10 Å to about2,000 Å before or after complexation with a metal-ion.

The enhancements of the present disclosure are applicable to othersubstrates including other glasses, metals, plastic substrates, such asTopas® COC substrates, available from TOPAS Advanced Polymers, Inc., andlike materials, or a combination thereof. Commonly owned and assignedcopending U.S. patent application Ser. No. 12/201,029, filed Aug. 29,2008, mentions plasma treated cyclic polyolefin copolymer surfaceshaving enhanced binding density for biologically active agents andcells. These plasma treated cyclic polyolefin copolymer surfaces may beselected as substrates in the present disclosure.

A Nb₂O₅ wave guide surface is well suited for biosensor-based cellassays, which can attach cells onto the bare Nb₂O₅ or nanoparticulatepolymer coated Nb₂O₅ biosensor surface, and have the associated cells inclose proximity of the detection zone of biosensor systems.

In embodiments, the disclosure provides a sensor and process forlabel-free detection having metal-chelate surface chemistry comprising alayer having polymer nanoparticles, that is, colloidal polymer particlesmade of a polymer having both metal chelating groups and ionizablegroups. In a second step after biomolecule immobilization, a chemicaltreatment step creates covalent interactions between the immobilizedbiomolecules and the biosensor surface, which precludes dissociation.

We have surprisingly discovered that chemistry based on polymernanoparticles bearing NTA groups and having a second covalent bondingstep between immobilized biomolecules and the substrate overcome theknown drawbacks by providing a sensor surface having a very high proteinimmobilization capacity and no protein dissociation, which makes thissensor surface highly compatible with low molecular weight drugscreening using LLD.

The metal-chelate surface having polymer nanoparticles can be easilyprepared by reaction between a maleic anhydride copolymer and a compoundbearing the chelating group, for example, the NTA. The NTA-modifiedmaleic anhydride copolymer can be obtained by reaction of the maleicanhydride copolymer and one reactant having at least one NTA group, atleast one thiol, at least one hydroxyl group, or at least one aminogroup, which group can react with the anhydride group leading to theformation of ester, thioester, imide, or amide group, respectively.Preferably, the compound can be selected from compounds described in EP0253303B1 (Hochuli, et al., “Neue Metallchelatharze”). More preferablythe compound is N,N bis-(Carboxymethyl)-L-Lysine, or its salt form suchas the N,N bis-(Carboxymethyl)-L-Lysine disodium salt mono hydrate, forexample.

The size of the NTA-maleic anhydride nanoparticles are preferably about5 to about 200 nm. The ratio between NTA groups and ionizable groups ofthe polymer backbone can be, for example, less than about 1 and greaterthan about 0.33. Thus, the ratio of NTA:COOH can be about 1:1 to about1:3.

In a second step, after biomolecule immobilization, treatment withEDC/NHS or like reagents, activates carboxylic groups that createcovalent bonds between affinity immobilized biomolecules and thesubstrate.

The disclosure provides an affinity chemistry with very highimmobilization capacity, which is important to detecting and observingbinding events. The surface chemistry of the disclosure provides a layerof polymer nanoparticles, which provides high protein/targetimmobilization. In an illustrative embodiment, the polymer EMA050NTA(i.e., an ethylene-maleic anhydride copolymer having about half or 50mol % of the anhydride moieties derivatized with NTA groups) was coatedon a gold chip, either: i) in 100% good solvent leading to 2D surfacechemistry (i.e., without particles); or ii) in mixed solvent leading to3D surface chemistry having nanoparticles. After further addition of thenickel solution to form chelated nickel, proteins immobilization wasperformed (FIG. 3). SPR responses clearly showed significantimprovement, such as greater than about double or more, for theimmobilization level when the coating included the polymernanoparticles. In addition, protein immobilization was made on a BiacoreNTA-chip and on a gold chip coated with the nanoparticle layer of thedisclosure using the same experimental conditions. SPR responses alsoindicated very high immobilization levels, such as double or triple theresponse, on the disclosed surfaces compared to a commercial chip (FIG.4). In addition, protein leaching appears to be significantly lower onthe surfaces of the disclosure compared to Biacore NTA-chip. Inembodiments, the disclosed articles and assay processes maintain highprotein immobilization even after an activation step with EDC/NHS.

In embodiments, the disclosure prevents protein dissociation usingcovalent attachment between immobilized target biomolecules and thesubstrate. In Example 2, his-tag carbonic anhydrase (II) was immobilizedon a nanoparticle polymer layer having NTA/Ni-ion complexes. When theEDC/NHS treatment was performed, no protein leaching was observedleading to high binding values with furosemide ligand. Conversely,without EDC/NHS treatment, interactions between proteins and thesubstrate are based only on affinity interactions and dissociation ofthe proteins from the surface was observed (FIG. 6), which reversibilityis an inherent aspect of affinity methods and consequently has nosignificant binding values (FIG. 5). In this example, it is important tonotice that using a high pH buffer for immobilization, such as whose pHwas higher than the pI of the proteins, had no impact on bindingresults.

Moreover, we have surprisingly discovered that performing the EDC/NHStreatment step is important for best results. In particular, it isimportant to distinguish methods of literature in which EDC/NHStreatment is applied before protein immobilization and the presentimmobilization method, which is accomplished in two steps. First,proteins are immobilized on the affinity surface referred to as, forexample, EMA050NTA-Ni, and secondly an EDC/NHS treatment is performed onimmobilized proteins. Example 3 shows the importance of this secondstep, as immobilization level and binding results are significantlyimproved compared to comparative methods (FIGS. 5 and 7, 8 and 10, and11 and 12).

Examples 4 and 5 show a specific aspect of protein immobilization onEMA050NTA-Ni chemistry as no significant immobilization levels wereobtained when non-tagged carbonic anhydrase was added on EMA050NTA-Nisurface (FIG. 9), and when histidine-tagged carbonic anhydrase was addedon the EMA050NTA surface without nickel treatment (FIG. 10).

In embodiments, the disclosure provides preparative methods for sensorsand biosensor surface layers having very high protein immobilizationcapacity, high observed protein immobilization, and a high bindingresponse.

The disclosed methods and sensors are straight forward and arecompatible with any label free detection platform, such as SPR, resonantgratings, Epic® sensor plates, or dual polarization interferometry, andoptionally including a micro-fluidic system.

The disclosure provides a method that permits immobilization of proteinsover a wide pH, especially at a pH level above the pI of protein target.

Referring to the Figures, FIG. 1A shows a schematic representation ofthe polymer of formula (I) comprising the nanoparticles. FIG. 1B wasdescribed above. FIG. 1C illustrates affinity capture, covalent bonding,and ligand capture steps.

FIG. 2A shows a SEM image of NTA derivatized EMA nanoparticles. FIGS. 2Band 2C show SEM images of EMA050NTA nanoparticles only, and EMA050NTAnanoparticles having been treated with ethanolamine blocking andnickel-ion, respectively.

FIG. 3 shows SPR responses for the surfaces with (310) and without (300)nanoparticle texture of Example 2.

FIG. 4 shows SPR responses using a commercially available NTA chip (400)compared with the present Example 3 (410).

FIG. 5 shows a comparison of His-tag CAII immobilization levels on anaffinity surface only (500, 510) and on the affinity/covalent surface ofExample 4 (520, 530).

FIG. 6 shows a comparison of protein leaching responses on the affinitysurface (600, 610) and the affinity/covalent surface of Example 4 (620,630).

FIG. 7 shows a comparison of binding values on the affinity surface only(700, 710) and on the affinity/covalent surface of Example 4 (720, 730).

FIG. 8 shows a comparison of His-tag CAII immobilization levels on theaffinity surface (800) and on the affinity/covalent surface (810 withNi²⁺ ion, 820 without Ni²⁺ ion) of a commercially available product andon the affinity/covalent surface of Example 5 (830).

FIG. 9 shows a comparison of protein leaching or dissociation responseson an affinity surface (900), on affinity/covalent surface of acommercially available product (910 with Ni ion, 920 without Ni ion),and on the affinity/covalent surface of Example 5 (930).

FIG. 10 shows a comparison of binding values on an affinity surface(1000), on an affinity/covalent surface of a commercially availableproduct (1010 with Ni ion, 1020 without Ni ion), and on theaffinity/covalent surface of Example 5 (1030).

FIG. 11 shows a comparison of CAII (i.e., CA2) or His-tag CAIIimmobilization levels on the affinity/covalent surface of Example 6(1100 his-tag CA2 in acetate; 1110 CA₂ in acetate; 1120 CA2 in HEPESplus salt; 1130 his-tag CA2 in HEPES plus salt).

FIG. 12 shows His-tag CAII immobilization levels on theaffinity/covalent surface of Example 7 (1200 EMA050NTA in acetate; 1210EMA050NTA in HEPES plus salt; 1220 EMA050NTA+Ni ion in acetate; 1230EMA050NTA+Ni ion in HEPES plus salt) with or without nickel iontreatment.

FIG. 13 shows four hour attachment human primary hepatocytes on EMA NTAsynthetic surface (as shown in FIG. 1B) relative to collagen I. Thesynthetic surface, EMA NTA supports attachment of human primaryhepatocytes similar to collagen I in serum culture conditions for 4hours.

FIG. 14 shows a comparison of attachment (24 h) and retention (7 d) ofhuman primary hepatocytes on EMA NTA synthetic surfaces (as shown inFIG. 1B) relative to collagen I; Synthetic EMA NTA surface supportsattachment of human primary hepatocytes (with serum and serum free)similar to collagen for 24 hours and 7 days.

FIG. 15 shows relative gene expression of three major metabolism genes(CYP450) in human primary hepatocytes cultured on EMA NTA syntheticsurface (as shown in FIG. 1B) relative to collagen I. The synthetic EMANTA surface shows a functional advantage (increase gene expression)relative to the biological surface, collagen I with serum (industrystandard). The serum free condition leads to an elevation of geneexpression for CYP2B6 and CYP3A4 on the EMA NTA surfaces.

FIG. 16 shows schematic representations of cells 1710 on a polymer layer1730 on a substrate 1720 alone and in the presence of a sandwich layer1740 which may be, for example, protein such as collagen, extracellularmatrix, Matrigel™ or a combination. sandwich layer which may be, forexample, protein such as collagen, extracellular matrix or Matrigel™ ora combination. Matrigel™ is a tumor cell extract made by BD (FranklinLakes, N.J.). Extracellular matrix refers to proteins that are presentin the extracellular spaces between cells. Extracellular matrix mayproteins such as collagen, elastin, fibronectins laminins andglycosaminoglycans.

FIG. 17A-C are micrographs of normal liver tissue (FIG. 17A), livercells grown on a collagen I coated substrate (FIG. 17B, on a coatedsubstrate as illustrated in FIG. 16A) and liver cells grown on acollagen I coated substrate, and overlaid with a Matrigel™ sandwichlayer (FIG. 17C, as illustrated in FIG. 16B) fluorescently stained toshow MRP2 transporter. MRP2 transporter is a marker of the bilecanaliculi structures at the apical domain between hepatocytes andindicates the degree of restoration of cell membrane polarity. FIG. 17Bshows bile canaliculi-like structures that appear like small dots ofstain (shown by white arrows). FIG. 17C shows extended and networkedbile canaliculi structures (shown by white arrows) indicating good (invivo like) restoration of cell polarity and a more in vivo-like cellmorphology. FIG. 17 shows that these bile caniliculi are present innormal liver tissue, in liver cells grown on a collagen I coatedsubstrate and in liver cells grown on a collagen I coated substrateoverlaid with a Matrigel™ sandwich layer.

FIGS. 18(A and B) are micrographs of liver cells grown on synthetic EMANTA surface alone (FIG. 18A), and in the presence of a Matrigel™sandwich layer (FIG. 18B) stained to show MRP2 transporter and to showthe presence of bile canaliculi structures (indicated by the whitearrows). The EMA NTA coated surface shows similar performance tocollagen. Without the Matrigel™ overlay (sandwich), bile canaliculi-likestructures appear as small dots of stain (shown by white arrow). Withthe Matrigel™ overlay extended lines of stain (as indicated by whitearrows) show restoration of polarity and bile canaliculi structures (invivo like morphology). Hepatocytes grown on the synthetic EMA NTAsurface with the Matrigel™ overlay (sandwich), exhibit hepatocytepolarity. In vivo-like morphology is restored to a greater degree thancells on other experimental surfaces (data not shown).

FIGS. 19(A and B) are micrographs of liver cells grown on synthetic EMANTA surface in the presence of a Matrigel™) sandwich in the presence ofserum (FIG. 19A) and in the absence of serum (FIG. 19B) stained to showMRP2 transporter. Hepatocyte polarity and morphological markers in serumand serum free culture conditions shows that both conditions supportcell culture and the development of bile canaliculi structures. Cellsmay be distributed more homogeneously across the EMA NTA surfaces in thepresence of serum. There may be more crowding and clustering of cells inthe serum free condition than in standard culture conditions with serum.Bile canaliculi structures appear to be further extended and moreinterconnected in the regions where cells are clustered under serum freeculture conditions. This may explain the slight increase in function(gene expression) observed in the serum free conditions. FIG. 19illustrates that the synthetic EMA NTA surface may provide a syntheticsurface which can be useful in culturing hepatocytes and other cells inthe absence of serum.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described disclosure, as well as to set forth the best modescontemplated for carrying out various aspects of the disclosure. It isunderstood that these examples in no way limit the scope of thisdisclosure, but rather are presented for illustrative purposes. Theworking examples describe how to prepare and use the LID sensor of thedisclosure which are contrasted with the comparative examples.

Example 1

Preparation of EMA-NTA (EMA050NTA) surface having preformednanoparticles 0.96 g of ethylene-alt-maleic anhydride copolymer (EMA)was dissolved in 100 mL of anhydrous DMSO under stirring for about 1hour. To the solution of previously prepared EMA was added 0.92 g ofbis-(CML), i.e., N,N′-bis[carboxymethyl]-L-lysine (nitriloacetic acid)(NTA) (available from Aldrich Chemical). The amount of NTA usedcorresponds to the amount required to convert about 50 mol % of theanhydride groups to NTA groups. The resulting solution was stirred for48 hours at room temperature. Then, this solution was diluted to about500 mL with DMSO and then 500 mL anhydrous IPA (50/50 v/v) was addedover about 1 hour with stirring to form polymer nanoparticles(EMA050NTA) characterized by dynamic light scattering (DLS) (about 30nm) and SEM (FIGS. 2B and 2C).

An NTA modified maleic anhydride copolymer (i.e., EMA-NTA), was obtainedin Example 1 by reaction of a maleic anhydride polymer, such asethylene-maleic anhydride copolymer (i.e., EMA), and a reactant havingat least one nitriloacetic acid group (such as NTA) and at least onethiol, hydroxyl group or amino group able to react with the anhydridegroup leading to the formation of the respective ester, thioester, imideor amide groups. The reactant can be selected, for example, fromcompounds described in EP 0253303B1. A preferred compound is, forexample, N″,N″-bis[carboxymethyl]-L-lysine (nitriloacetic acid) (NTA),also known as bis-CML, or salts thereof, such as the N,Nbis-(Carboxymethyl)-L-Lysine disodium salt mono hydrate. Other names forNTA include, for example, AB-NTA; aminobutyl-NTA;N-(5-Amino-1-carboxypentyl)iminodiacetic acid.

A 5 wt % solution of aminopropylsilane (APS) in DI water was preparedfrom the 20 wt % commercially available solution. Then, 384-well Epic®insert was dip coated with an aliquot of the APS solution for 10 min.After incubation for 2 to about 24 hours, the APS solution was removed,rinsed three times with water, and three times with anhydrous ethanol,then dried with a stream of nitrogen.

After drying, the APS pre-coated insert was incubated for 10 min. atroom temperature in the suspension of EMA050NTA nanoparticles previouslyprepared. Then, the insert was rinsed with ethanol and was dried bymeans of a gentle nitrogen stream.

Example 2

SPR responses on surfaces with and without nanoparticle texture Coatingthe EMA050NTA of the disclosure on gold chip: A solution of3-mercaptopropyl) trimethoxysilane (3-MPT, Aldrich Chemicals) at 20 mMin anhydrous ethanol is added on cleaned gold chip during 2 hours atroom temperature. After washing, a solution of 5%aminopropylsilsesquioxane oligomer (APS, Gelest) is added on chip during15min at room temperature. Then, the precoated chip was contacted for 10min at room temperature with EMA polymer in 100% NMP (2D strategy), andanother chip was contacted for 10 min at room temperature with acolloidal solution of EMA polymer dispersed NMP/IPA (90/10) (3Dstrategy). Then, a solution of CML (0.5 g in 10 mL of water) was addedonto both chips for 1 hour at room temperature. The addition of a nickelsolution was made on Biacore X equipment. His-tag carbonic anhydrase wasimmobilized on these two surfaces (2D and 3D) at 100 μg/mL in Tris, HCl20 mM (FIG. 3).

Example 3

SPR responses using a commercially available NTA chip compared with anNTA nanoparticulate modified surface The procedure of Example 2 wasrepeated to prepare a gold chip with a coating made from colloidalsolution of the EMA polymer. Addition of the nickel solution was made inBIACORE X equipment on an experimental chip and on a commercial BIACORENTA chip. His-tag carbonic anhydrase was immobilized on these twosurfaces at 125 μg/mL in Tris, HCl 20 mM (FIG. 4).

Example 4

Epic® analysis using an affinity surface only modification compared to“affinity and covalent” surface modification The procedure of Example 1was repeated to prepare 384-well microplate coated with EMA050NTA.Different approaches were performed on this polymer layer (microplatedivided in four areas). Part 1 and part 4 were in contact with a nickelsolution. Then, His-tag carbonic anhydrase was immobilized at 50 μg/mLin acetate overnight on both parts. In a second step, after washing,EDC/NHS (200 mM/50 mM) treatment was applied only on part 4. Concerningpart 2, EDC/NHS treatment was performed on EMA050NTA before nickeladdition. Concerning part 3, nickel addition was made before EDC/NHStreatment. Then, His-tag carbonic anhydrase was immobilized at 50 μg/mLin acetate overnight on parts 2 and 3 (Epic® responses, FIG. 5).Addition of buffer PBS+0.1% DMSO was performed on the immobilizedproteins of a first part of the microplate and protein leaching wasmeasured on Epic® equipment (FIG. 6). On another part of the microplate,furosemide ligand was added at 10 μM in PBS and 0.1% DMSO on immobilizedproteins and the binding values were measured with Epic® equipment (FIG.7). The immobilization of his-tag CA2 was accomplished in two buffers(acetate pH 5.5 or HEPES pH 7.4). Results showed immobilization levelsgreater than about 5,000 pm (FIG. 5) and binding greater than 25 pm(FIG. 7). The loss of protein was estimated in both instances to beabout 3 pm, or less than about 0.1 percent of the immobilized protein.

Example 5

Epic® analysis using a known “affinity and covalent” approach comparedto the present disclosure The procedure in Example 1 was repeated toprepare 384-well microplate coated with EMA050NTA nanoparticulates.After addition of the nickel solution, His-tag carbonic anhydrase wasimmobilized at 50 μg/mL either in acetate buffer or in HEPES and salt(i.e.,150 mM NaCl) buffer overnight on inserts. After washing, anEDC/NHS treatment (200 mM/50 mM) was performed during 30 min in water,protein immobilization levels were obtained using Epic® equipment (FIG.8). Addition of buffer (PBS and 0.1% DMSO) was performed on immobilizedproteins of a part of microplate and protein leaching was measured onEpic® equipment (FIG. 9). On the other part of the microplate,furosemide ligand was added at 10 μM in PBS and 0.1% DMSO on immobilizedproteins and binding values were measured with Epic® equipment (FIG.10).

Example 6

CAII or his-tag CAII immobilization level on affinity surface Theprocedure of Example 1 was repeated to prepare a 384-well microplatecoated with EMA-NTA nanoparticulates. After addition of the nickelsolution, His-tag carbonic anhydrase and non-tagged carbonic anhydrasewere immobilized at 50 μg/mL in either acetate or in HEPES and 150 mMNaCl buffers overnight on inserts. After washing, an EDC/NHS treatment(200 mM/50 mM) was performed over 30 min in water, and immobilizationlevels were obtained using Epic® equipment (FIG. 11).

Example 7

His-Tag CAII Immobilization on Affinity Surface With and Without NickelTreatment

The procedure of Example 1 was repeated to prepare a 384-well microplatecoated with EMANTA nanoparticulates. Addition of the nickel solution wasperforated only on half of the microplate. His-tag carbonic anhydrasewas then immobilized at 50 μg/mL, in either acetate buffer alone or inHEPES and 150 mM NaCl buffer overnight on each part of the inserts.After washing, an EDC/NHS treatment (200 mM/50 mM) was performed over 30min in water and immobilization levels were obtained using Epic®equipment (FIG. 12).

Example 8

Cell Culture

Human primary hepatocytes were plated with serum containing (orserum-free) MFE plating medium (in house media preparation, similar tomedia commercially available from XenoTech) and cultured at 37° C., in ahumidified atmosphere of 5% CO2. Cells are maintained in serum-free MFEmaintenance medium (in house media preparation, similar to mediacommercially available from XenoTech) and microscopically observed dailyto monitor cell culture health and morphology. Primary hepatocytes wereseeded (plated) on the surface at 60,000 cells in 100 uL media per wellin 96 well microplate format. Media was replaced daily. HepG2/C3A cells(ATCC #CRL-10741) were cultured in Eagle's Minimum Essential Medium(ATCC #30-2003) optionally supplemented with 10% Fetal Bovine Serum(Invitrogen #16000-077) and 1% Penicillin-Streptomycin (Invitrogen#15140-155). Cells were incubated at 37° C., in 5% CO2 and 95% relativehumidity. C3A cells were seeded on the polymer-coated substrates at 5000cells in 100 uL media per well in 96 well microplate format. Media wasreplaced daily. Number of cells in culture on EMA NTA surface wasquantified using Cell-Glo® Luminescent Cell Viability Assay (PromegaG7571). Results are shown in FIGS. 13 and 14.

Example 9

Assessment of Hepatocyte Function via Gene Expression

After 7 days of culture, cells were lysed to isolate RNA using RNeasy®Mini Kit (Qiagen, Catalog #74106(250), RNase-Free DNase Set (Qiagen,Catalog #79254) and Quant-iT™ RiboGreen® RNA Reagent and Kit(Invitrogen, Catalog #R11490) and the endogenous gene expression of CYP1A2, 2B6, and 3A4 genes were assessed via Quantitative Real-Time PCR andTaqMan® Low Density Arrays on day 7 of culture, see FIG. 15.

Example 10

Assessment of Morphological Markers

Cell polarity using MRP2 morphological marker for apical domain (bilecanaliculi) was assessed at day 7 via immunochemical assays. Fluorescentmicroscopy was used to image the fluorescently stained markers thatindicate the presence of MRP2 in the apical domain as an indication ofbile canaliculi structures (FIGS. 17, 18 and 19). A Matrigel™ overlaywas added to samples after 1 day in culture to provide a EMANTA/Matrigel™ sandwich culture configuration and demonstrated extensionof bile canaliculi structures as evidence of change in morphology andrestoration of membrane polarity. This phenomenon is observed incollagen I/Matrigel™ sandwiches and further demonstrates that thesynthetic EMA NTA surface can be used to replace non-synthetic orbiological surfaces.

The disclosure has been described with reference to various specificembodiments and techniques. However, it should be understood that manyvariations and modifications are possible while remaining within thespirit and scope of the disclosure.

1. A cell culture article comprising: a substrate having nanoparticles(NP) on the substrate surface, the nanoparticle comprises: a polymer offormula (I)

having at least one of: a metal-ion chelating group (x), an ionizablegroup (y), and a surface substantive group (z), where R is hydrogen or asubstituted or unsubstituted, linear or branched, monovalent hydrocarbylmoiety having from 1 to 6 carbon atoms; R′ is a substituted orunsubstituted, linear or branched, divalent hydrocarbyl moiety resultingfrom copolymerization of an unsaturated monomer having from 2 to 18carbon atoms with maleic anhydride; R″ is a substituted orunsubstituted, linear or branched, divalent hydrocarbyl moiety havingfrom 1 to 20 carbon atoms; S comprises at least one point of attachmentto the substrate; W comprises at least one bi-dentate group; X is an—NH—, —NR—, or O; the mole ratio of x:(y+z) groups is from about 2:8 toabout 8:2; and the nanoparticles have a diameter of from about 10 toabout 100 nanometers.
 2. The article of claim 1 wherein R is a hydroxysubstituted, monovalent hydrocarbyl moiety having from 2 to 6 carbonatoms.
 3. The article of claim 1 wherein the mole ratio x:(y+z) is about1:1.
 4. The article of claim 1 wherein S comprises at least one of: ametal oxide, a mixed metal oxide, a polymer, a composite, or acombination thereof; a surface modified substrate; or a combinationthereof.
 5. The article of claim 1 wherein R is a hydroxy substitutedalkyl having from 2 to 4 carbon atoms; R′ is a divalent hydrocarbylmoiety having from 2 to 10 carbon atoms; R″ is a substituted orunsubstituted, divalent hydrocarbyl moiety having from 3 to 6 carbonatoms; S is an aminosiloxane treated glass or plastic substrate; Wcomprises at least one iminodiacetic acid, nitrilotriacetic acid,triazacyclononane, aminoethylethanolamine, triethylenetetramine,2-hydroxypropane-1,2,3-tricarboxylate, or a mixture thereof; X is —NH—;the mole ratio x:(y+z) is from about 2:1 to about 1:2; and thenanoparticles have a diameter of from about 10 to about 100 nanometers.6. The article of claim 1 wherein the nanoparticles (NP) on thesubstrate surface comprise a layer of nanoparticles, a polymer film, ora combination of nanoparticles and polymer film.
 7. The article of claim6 wherein the layer or film comprises incomplete substrate surfacecoverage.
 8. (canceled)
 9. The article of claim 1 wherein R is a hydroxysubstituted alkyl having from 2 to 4 carbon atoms; R′ is a divalenthydrocarbyl moiety having from 2 to 10 carbon atoms; R″ is a substitutedor unsubstituted, divalent hydrocarbyl moiety having from 3 to 6 carbonatoms; S is a plastic substrate; W comprises nitrilotriacetic acid; X is—NH—; the mole ratio x:(y+z) is from about 2:1 to about 1:2; and thenanoparticles have a diameter of from about 10 to about 100 nanometers.10-18. (canceled)